Carry-over problems

Hello,I am currently working on a new UPLC/MS method with 4 analytes. I am having carry-over problems with one of the compounds (the other three have no or neglible carry-over).When a series of blanks was injected after a high calibration standard, carry-over peak with areas approximately 0.3-0.5%, 0.08%, 0.04%, 0.03%, 0.02% of the standard are observed. The carry-over peak area in the initial blank injection is higher than the lowest standard.Initially I was using injection mode PLNO, 25µL inj vol with 50 µL loop. I increased the needle wash volumes to 1000/1500 µL. I tried changing from PEEK needle to SS needle with no significant difference.I have tried various strong needle washes - higher organic, lower organic, higher pH, acidic pH - no significant improvement.I did see slight improvement after changing injection mode to PL(PA) then to Full Loop (20µL loop) (decrease in 1st blank from ~0.5% to ~0.3%).Method conditions are: MP A= 0.1% Formic Acid, MP B = 1:1 ACN:MeOH, WNW = 90:10 Water:ACN, SNW = 2:2:1 ACN:MeOH:Water with 5% formic acidGradient 10-95%B in 1.5 min, hold 95%B until 2 min, then re-equil to 4 min. Flow 0.4 mL/min. There was no significant difference btwn using load ahead (offline 3.0 min) vs no load ahead. Samples are dissolved in 20:80 ACN: aq Formic Acid (no difference in carry-over btwn 0.1, 0.5 or 1%).Peak with carry-over elutes at ~1.3 min.Any suggestions to solve this problem would be greatly appreciated.Thank you.

I have a few questions about your method. We should consider one at a time to see what helps. Change one at a time and go back to original if it does not help.

(1) In your data you have not mentioned the column dimensions. A 25 µL injection volume would be rather large for a typical 2.1 mm ID, sub 2µm column. However, the flow rate, at 400 µL per minute does suggest a UPLC column. Can you confirm the column’s dimensions.

(2) My concern is that are making a 25 µL injection on a column that has a pretty low volume. I would like to know the concentration of your compounds, we may need to inject less. I am also a little concerned that the carryover may lead to contamination of the system – can you get a clean blank now? If not we need to clean up the system first. 2 µL volume for a sub 2µm column would be more appropriate.

(3) Back to column dimensions – depending on the volume your column the re-equilibration time could be lengthened – rule of thumb says x5 column volumes, so for a 2.1 x 50 mm column you need over a 1,000 µL, your method at 2 min re-equil might be too short. I would lengthen the time by x3 o 4 to see what the effect is.

(4) Back to your sample – like maniarni I wonder about solubility issues, as the carryover is selective. Do you think your compound is soluble in 10% aqueous where the gradient starts? The sample diluent is 20% organic, which suggests that it is more organic rather than less – so I would start that gradient at 20% rather than 5%.

(5) The fact that you see a carryover difference in injection modes is not a surprise, each modes uses a completely different mechanism and so carryover can differ. Generally, the rule is FL is best, then PAPL and PLNO last for carryover. With PLNO mode in order to get rid of carryover, rather than simply increase the wash volumes alone, again as maniarni says, what is that compound most likely to be solubilized in? Is it polar, non-polar, acidic, basic etc? As needlewash is not very critical in PLNO we can make the weak and strong wash both very strong and with different selectivities. We could put Formic in both for example, we can add in DMSO in the mix too.

(6) Also if the compounds prefer organic with PLNO, I would strengthen the sample diluent as much as possible – make it very organic, as much as the chromatography will take, if that’s what your samples prefer. And here injecting less would assist too.

(7) The strong wash is 90% organic and your gradient goes to 95% - so I would make it even more organic, but as I do not know the nature of the analytes, I cannot tell exactly what would really be best, but 95% anyway, which corresponds to where the gradient finishes. We often add IPA to our SW mix. In PLNO, it has the VVD is used, for larger injection volumes sample can diffuse to where it should not go. Both FL and PLPA do not use the volume detection device, there is a possibility that the compound could be could be sticking there, but I would ensure that you check the other parameters I raised first. If the needle change makes no difference then the needle is not the issue.

These are some things to think about, but please get us the concentration of the sample and the column dimensions, can we also see a chromatogram. It would be helpful to know which MS you are using as well.

Hope this helps, and any data you provide I will run by our best chemists!

While carryover is most typically associated with the injector, another thing to consider is that the analyte in question is not completely eluting from the column and appears in subsequent blank injections. The fact that its retention time is 1.3 minutes tells us it requires a high organic concentration to elute (~ 75 % . The time for your strong solvent flush of the column is extremely short (0.5 minutes). At your flow rate of 0.4 mL/min this translates to 200 uL. You do not mention the length of your column but even for a short 50 mm column this is less than 2 column volumes (liquid volume of 2.1 x 50 mm ACQUITY column is ~ 115 uL). For UPLC, the number of column volumes that are recommended for strong solvent flushing and column re-equilibration are no different than those for HPLC, namely 5 - 10 column volumes. Thus a more appropriate strong solvent flush time would be 1.5 minutes for a 50 mm column at a flow rate of 0.4 mL/min.

Before you investigate further with wash chemistry, you might give this a go.

1) I was using an Acquity HSS T3 1.8µm 2.1x50 mm column, then I switched to a Halo C18 2.7 µm, 2.1x100 mm column and found I had less carry-over.

2, 6) Injection volume is high because concentrations are low. Upper concentration is approx 8 ng/mL soln conc. The concentrations are low because I am trying to decrease the range on an existing method without changing the sample preparation steps. I have also decreased the amount of organic in the final extract compared to the existing method so that I could inject more without having terrible peak shape.

3) I will try increasing the re-equilibration time. I did increase it when I switched to the longer column, but probably not by enough.

4,5) The compounds are relatively hydrophilic. The standard dissolves easily and is stable in 15% ACN in aqueous dilute acid. Changing the gradient start to 20% did decrease the carry-over.

7) I tried SNW 1:1:1:1 IPA:ACN:MeOH:Water + 2% formic acid which decreased the amount of carry-over that I was seeing. Even better is 3:1 IPA:Water + 2%formic acid.

With the column change, gradient change and IPA in the SNW using either full loop or partial loop (not PLNO), I am now seeing approx 0.1% peak area of the standard in the first blank injection and baseline noise by the second injection. I have also learned a lot more about the different injection modes on the UPLC.